Gram-negative bacteria have an outer membrane comprised of components including proteins, lipoproteins, phospholipids, and glycolipids. The glycolipids comprise primarily endotoxin-lipopolysaccharides (LPS) or lipooligosaccharides (LOS), depending on the genus of bacteria. LPS are molecules comprised of    a) a lipid A portion which consists of a glucosamine disaccharide that is substituted with phosphate groups and long chain fatty acids in ester and amide linkages;    b) a core polysaccharide which is attached to lipid A by an eight carbon sugar, KDO (ketodeoxyoctonoate), and heptose, glucose, galactose, and N-acetylglucosamine; and    c) an O-specific side chain comprised of repeating oligosaccharide units which, depending on the genera and species of bacteria, may contain mannose, galactose, D-glucose, N-acetylgalactosamine, N-acetylglucosamine, L-rhamnose, and a dideoxyhexose (abequose, colitose, tyvelose, paratose, trehalose). LOS has a similar structure as LPS, containing a lipid A portion and a complex carbohydrate structure, but differs in that it does not contain repeating O-side chains.
The major antigenic determinants of gram-negative bacteria are believed to reside in the carbohydrate structure of the O-specific side chain of LPS and the complex carbohydrate structure of LOS. These carbohydrate structures may vary for different species of the same genera of gram-negative bacteria by varying one or more of the sugar composition; the sequence of oligosaccharides; the linkage between the oligosaccharides; and substitutions/modifications of an oligosaccharide (particularly a terminal oligosaccharide).
LPS and LOS have been considered as bacterial components which have potential as vaccine immunogens because of the antigenic determinants (“epitopes”) residing in their carbohydrate structures. However, the chemical nature of LPS and LOS prevent the use of these molecules in vaccine formulations; i.e., active immunization with LPS or LOS is unacceptable due to the inherent toxicity of the lipid A portion. The pathophysiologic effects induced (directly or indirectly) by lipid A of LPS or LOS in the bloodstream include fever; leucopenia; leucocytosis; the Shwartzman reaction; disseminated intravascular coagulation; abortion; and in larger doses, shock and death. Accordingly, there are no currently available vaccines which induce antibody responses to LPS or LOS epitopes.
As shown in FIG. 1, the lipid A portion of endotoxin generally comprises a hydrophilic backbone of glucosamine disaccharide which is either monophosphorylated or diphosphorylated (positions 1 and 4′); and which carries at least six molecules of ester- and amide-bound fatty acids. Four molecules of (R) 3-hydroxytetradecanoate (e.g. 3-hydroxymyristoyl or β-hyroxymyristic acid or β-OH) are linked directly to the lipid A backbone at positions 2, 3, 2′, and 3′. Hydroxyl groups of two of the four molecules of β-OH are substituted with normal fatty acids (termed “secondary acyl chains”, and including dodecanoate, tetradecanoate, and hexadecanoate) in forming acyloxyacyl groups.
One approach to making a detoxified endotoxin molecule involves isolating the endotoxin, and enzymatically-treating the isolated endotoxin with a human neutrophilic acyloxyacyl hydrolase (U.S. Pat. Nos. 4,929,604, 5,013,661 and 5,200,184). The acyloxyacyl hydrolase hydrolyzes the fatty acids (non-hydroxylated, secondary acyl chains) from their ester linkages to hydroxy groups of β-OH (hydroxylated). The resultant altered endotoxin, from enzymatic treatment, contained a lipid A moiety lacking non-hydroxylated fatty acids. This altered endotoxin exhibited reduced in vivo toxicity, but retained antigenicity.
Another approach involves a method of modifying isolated endotoxin by selectively removing the β-OH that is ester-linked to the reducing-end glucosamine backbone at position 3 (U.S. Pat. No. 4,912,094; Reexamination B1 4,912,094). The selective removal of β-OH was accomplished using alkaline hydrolysis. The resultant modified endotoxin exhibited reduced in vivo toxicity, but retained antigenicity.
Both approaches involve chemically treating isolated endotoxin. Neither approach discloses the production in a gram negative bacterial pathogen of an endotoxin having substantially reduced toxicity, yet retaining antigenicity. Further, there has been no disclosure of the use of a gram-negative bacteria, which has been engineered to produce an endotoxin having substantially reduced toxicity and yet retaining antigenicity, in a prophylactic or therapeutic vaccine against endotoxic shock and gram-negative bacteremia.